AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. In clinical trial, rAAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) diseases and patients are able to regain normal vision after receiving the vectors. However, its use in other genetic diseases such as hemophilia still face considerable challenge. Systemic delivery of rAAV into human subjects would require a lot more vectors than targeting the retina tissue. Prelimary studies have demonstrated more than 99.9% of rAAV vectors were wasted in the varous stages of rAAV transduction. In addition, rAAV genomes packaging preference remains an unsettling issues. In order to further develop recombinant AAV for human gene therapy, we hypothesize that it would eliminate unwanted replication competent AAV particle formation and improve rAAV production by sequetering the helper function and vector sequencing into different cellular compartment. Advanced genomics and proteomics will be introduced to study rAAV packaging preference and encapsidation mechanism. Finally, we identified that cytoplasm is major site for rAAV genome loss. An innovative assay will be established to track rAAV genomes in cytoplasm host and mechanism will be studied to reduce rAAV genomes in cytoplasm of the host cells. Hence, our three specific aims are 1. To develop and optimize the next generation of rAAV packaging system~ 2. To characterize rAAV genome integrity and rAAV packaging mechanisms~ 3. To study the molecular status of recombinant AAV genomes in the cytoplasm. Completion of specific aims in this application will markedly enhance the way of rAAV vectors to be used in the human gene therapy field.

Public Health Relevance

The completion of this project may provide a solution for producing high quality rAAV vector economically. Our work may help design optimized vector for human gene therapy and improve the quality of life of patients with genetic diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BST-N (90))
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Skarlatos, Sonia
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Temple University
Schools of Medicine
United States
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Dong, Biao; Duan, Xunbao; Chow, Hoi Yee et al. (2014) Proteomics analysis of co-purifying cellular proteins associated with rAAV vectors. PLoS One 9:e86453
Dong, Biao; Moore, Andrea R; Dai, Jihong et al. (2013) A concept of eliminating nonhomologous recombination for scalable and safe AAV vector generation for human gene therapy. Nucleic Acids Res 41:6609-17
Kapranov, Philipp; Chen, Lingxia; Dederich, Debra et al. (2012) Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing. Hum Gene Ther 23:46-55
Lu, Hui; Qu, Guang; Yang, Xiao et al. (2011) Systemic elimination of de novo capsid protein synthesis from replication-competent AAV contamination in the liver. Hum Gene Ther 22:625-32
Dong, Biao; Nakai, Hiroyuki; Xiao, Weidong (2010) Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 18:87-92
Chen, Lingxia; Lu, Hui; Wang, Jinhui et al. (2009) Enhanced factor VIII heavy chain for gene therapy of hemophilia A. Mol Ther 17:417-24
Lu, Hui; Chen, Lingxia; Wang, Jinhui et al. (2008) Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette. Hum Gene Ther 19:648-54
Wang, Jinhui; Xie, Jing; Lu, Hui et al. (2007) Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction. Proc Natl Acad Sci U S A 104:13104-9